Carbohydrate moieties of glycoconjugates are involved in numerous important biological processes. Sharon, N. (1984) Trends Biochem. 9, 198-202; Feizi, T. & Childs, R. A. (1985) Trends Biochem. Sci. 10, 24-29; Feizi, T. Childs, R. A. 91987) Biochem. J. 245, 1-11. In addition to their metabolic and storage roles, carbohydrates are covalently attached to numerous other molecules such as proteins and lipids. Molecules such as glycoproteins and glycolipids are generally referred to as glycoconjugates. The biological importance of the carbohydrate portion of glycoconjugates can be seen, for example, in the role they play in affecting the ability of glycoproteins to perform their biological functions, including such functions as ligand or receptor recognition.
As a consequence of their diverse and important biological functions, aberrations in the synthesis, degradation, or modification of carbohydrates may give rise to several diseases. Similarly many diseases may alter the body's physiology so as to give rise to altered carbohydrate metabolism or the improper glycosylation of proteins, lipids and other glycoconjugates in the body.
Many of the biologically active carbohydrates in the body are polysaccharides and oligosaccharides that are produced in a variety of related forms rather than having a single defined structure. These families of related carbohydrates are frequently found to be components of the same glycoprotein. These families of glycoproteins that share the same polypeptide structure, but display variation in the glycosylation pattern have been referred to as glycoforms, Rademacher, et al, Ann. Rev. Biochem., 57:789-838 (1988).
The relative abundance of members of glycoform family members has been shown to vary in accordance with certain disease states. For example, the disfibrinogenemia associated with liver disease has been associated with variations in the glycosylation of fibrinogens, Martinez, J., et al, Blood, 61:1196-202 (1983), and rheumatoid arthritis has been associated with changes in glycosylation of IgG, Parekh et al, Nature, 316:452-457 (1985).
Diseases based on improper metabolism of carbohydrates from glycoconjugates are well known. The general category of diseases is known by a variety of names, including lysosomal storage disorders, heteroglycanoses, inborn errors of complex carbohydrate metabolism, mucopolysaccharidoses and others. Each of these diseases is the result of a genetic inability to produce one or more of the enzymes required for the stepwise degradation of glycoproteins, mucopolysaccharides or glycolipids, or the carbohydrate portion of said glycoconjugates.
When one of these enzymes in the degradation pathway is incorrectly produced or missing completely, the molecule produced in the last working step of the degradation pathway accumulates due to the body's inability to further cleave the molecule. Over time, the compound that cannot be degraded accumulates to such an extent that it impedes normal biological function in a wide variety of cells throughout the body.
The consequences of this type of genetic defect vary among the different enzyme deficiencies, but the symptoms of these diseases may include organomegaly, corneal opacities, skeletal abnormalities and progressive mental retardation.
Thus, it is of interest to provide a general technique for the diagnosis of a variety of diseases characterized by altered levels of carbohydrates in which the diagnostic technique does not require an a priori detailed knowledge of the structure of the carbohydrates.
In order to accomplish this objective, it is necessary to develop a means for detecting carbohydrates, quantitating them and determining their chemical structures. Attempts to elucidate carbohydrate structure have depended on purification techniques such as high performance liquid chromatography (h.p.l.c.) and other chromatographic methods. Subsequent structural analysis has been performed by a combination of classical derivatization and degradation procedures, mass spectrometry (m.s.) and nuclear magnetic resonance (n.m.r.). McNeil, M., Darvill, A. G., Aman, P., Franzen, L. E. & Albersheim, P. (1982) Methods Enzymol. 83, 3-45; Barker, R., Nunez, H. A., Roseyear, P. & Serianni, A. S. (1982) Methods Enzymol. 83, 58-69; White, C. A. & Kennedy, J. F. (1986) in Carbohydrate Analysis, A Practical Approach (Chaplin, M. F. & Kennedy, J. F., eds.). pp. 37-54, IRL Press, Oxford; Welply, J. K. (1989) Trends Biotechnol. 7, 5-10. Although these are powerful methods, they have significant limitations. The quantities of material required for some analyses can be relatively large compared with that which is available from many specific biological sources. In addition, the equipment required for these methods is expensive and requires considerable expertise and technical support, which tends to restrict their use to few laboratories.
In order to overcome some of these disadvantages, several workers have used specific glycosidases to degrade complex oligosaccharides and have deduced their structures after separating the degradation products by various chromatographic and electrophoretic techniques. Wang, W. T., LeDonne, Jr., N. C., Ackerman, B. & Sweeley, C. C. (1984) Anal. Biochem. 141, 366-381; Welply, J. K. (1989) Trends Biotechnol. 2, 5-10; Tomiya, N. Kurono, M., Ishihara, H. Tejima, S., Endo, S., Arata, Y. & Takahashi, N. (1987) Anal. Biochem. 163, 489-499; Wenn, R. V. (1975) Biochem. J. 145, 281-285; Montreuil, J. Bougqulet, S., Debray, J. Pournet, B., Spik, G. & Strecker, G. (1986) in Carbohydrate Analysis: A Practical Approach (Chaplin, M. F. & Kennedy J. F., eds.), pp. 143-204, IRL Press, Oxford; Kobata, A. (1979) Anal. Biochem. 100, 1-14; Tarentino, A. L. Trimble, R. B. & Plummer, Jr., T. H. (1989) Methods Cells Biol. 32, 11-139. This type of analysis can be used with picomolar quantities of material. In order to enable the sensitive detection of such quantities, a number of methods have been described in which saccharides and glycopeptides have been labelled with either .sup.3 H, chromophores or fluorophores and the derivatives separated either chromatographically or electophoretically. Weitzman, S. Scott, V. & Keegstra, K. (1979) Anal. Biochem. 97, 438-449; Wang, W. T., LeDonne, Jr., N. C., Ackerman, B. & Sweeley, C. C. (1984) Anal. Biochem. 141, 366-381; Hase, S., Ikenaka, T. a Matsushima, Y. (1979) J. Biochem. (Tokyo) 85, 989-994; Prakash, C. & Vijay, I. A. (1983) Anal. Biochem. 128, 41-46; Tomiya, N. Kurono, M., Ishihara, H. Tejima, S., Endo, S., Arata, Y. & Takahashi, N. (1987) Anal. Biochem. 163, 489-499; Wenn, R. V. (1975) Biochem. J. 145, 281-285; Narasimhan, S., Harpaz, N., Longmore, G. Carver, J. P., Grey, A. A. & Schachter, H. (1980) J. Biol. Chem. 225, 4876-4884; Hase, S., Ibuki, T. & Ikenaka, T. (1984) J. Biochem. (Tokyo) 95, 197-203; Poretz, R. D. & Pieczenik, G. (1981) Anal. Biochem. 115, 170-176; Das, O. P. & Henderson, E. J. (1986) Anal. Biochem. 158, 390-398; Towbin, H., Schoenenberger, C. A., Braun, D. G. & Rosenfelder, G. (1988) Anal. Blochem. 173.1-9; Maness, N. O. & Mort, A. J. (1989) Anal, Biochem. 178, 248-254; Hara, S., Yamaguchi, M. Takemori, Y., Furuhata, K., Ogura, H. & Nakamura, M. (1989) Anal. Biochem. 179, 162-166; Honda, S., Iwase, S., Makino, A. & Fujiware, S. (1989) Anal. Biochem. 176, 72-77.
Similarly, the diagnosis of carbohydrate metabolism disorders has historically been difficult because few methods exist for the separation, detection and identification of a wide variety of complex carbohydrates. The two main methods that have been employed are carbohydrate staining techniques and chromatographic separation and detection methods.
The carbohydrate staining techniques, including the Berry Spot Test and the dimethylmethylene blue (DMB) assay rely on a specific reaction between a chemical dye and a specific class of oligosaccharides. The major application of these methods have been with the mucopolysaccharidoses, which are disorders of glycosaminoglycan degradation. These tests have been proposed for large scale screening, but they are limited to the specific disorders for which the chemistry is designed, and the tests have had a problem with a large number of false positive diagnoses. Sewell, et al, Klin Wochenschr, 57:581-585 (1979), or Lurincz, et al, Ann. Clin. Lab. Sci., 12:258-266 (1982).
Chromatographic separation of oligosaccharides from glycoconjugates has also been proposed as a screening technique for these diseases, but there is not one chromatographic technique or set of chromatographic conditions that will facilitate the separation of the range of carbohydrate-based compounds that accumulate in all of these diseases. Thin layer chromatography (TLC), high performance liquid chromatography and gas chromatography have some utility in the diagnosis of the carbohydrate metabolic diseases, but they have found limited acceptance in clinical laboratories as a result of their limitations and/or complexity.
A new type of procedure has been described in which reducing saccharides are derivatized with fluorophores and the derivatives separated by one-dimensional polyacrylamide gel electrophoresis (PAGE). The fluorophore assisted carbohydrate electrophoresis technique is described in detail in U.S. Pat. Nos. 4,874,492 and 5,104,508, which are herein incorporated by reference. Fluorophore assisted carbohydrate electrophoresis permits the electrophoretic separation of a complex mixture of carbohydrates into distinct bands on a gel. Prior to electrophoresis, a carbohydrate mixture for analysis is treated with a fluorophore label that combines with the reducing end of the carbohydrates for analysis. The fluorophore label permits the quantitative measurement of the labeled carbohydrates by fluorescence. The fluorophore label either is charged or coupled with a charge imparting species when the fluorophore itself is uncharged. Thus the label not only fluorescently tags the carbohydrates, but imparts an ionic charge, permitting hitherto uncharged carbohydrates to migrate in an electric field.
After the carbohydrates have been labeled, the sample is subsequently subjected to polyacrylamide gel electrophoresis, or similar electrophoresis separation means, in order to separate and concentrate the labeled carbohydrates into bands. The separated carbohydrates may be visualized directly by photoelectric menus fluorescence under U.V. light and the banding patterns stored photographically. Alternatively the separated carbohydrates may be visualized by photoelectric means, including laser-scanner photomultiplier tube systems and cooled charge coupled devices (CCD). CCD's are semiconductor imaging devices that permit the sensitive detection of emitted light. CCDs and their uses are described in U.S. Pat. Nos. 4,874,492 and 4,852,137 which are herein incorporated by reference. The image produced by the CCD may be subsequently transferred to a computer wherein the bands may be analyzed with respect to intensity, mobility, standards, and the like.
When performing fluorophore assisted carbohydrate electrophoresis diagnosis, electrophoretic separation should take place to an extent sufficient to independently resolve bands of diagnostic carbohydrates specific for the disease of interest. Electrophoresis may proceed past the point where some carbohydrates have been removed from the electrophoresis separation medium.
Suitable fluorescent labels for use in fluorophore assisted carbohydrate electrophoresis include 8-aminonapthalene-1,3,6-trisulphonic acid (ANTS), 1-amino-4-napthalene sulfonic acid (ANSA), 1-amino-6,8-disulphonic acid (ANDA), lucifer yellow and 2-aminoacridone. The present invention is particularly directed toward fluorophore assisted carbohydrate electrophoresis wherein 2-aminoacridone is employed as the fluorescent labelling reagent.